Shanghai Veterinary Research Institutegrid.464410.3, Chinese Academy of Agricultural Sciences (CAAS), Shanghai, China.
Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu, China.
Appl Environ Microbiol. 2022 Jun 14;88(11):e0240921. doi: 10.1128/aem.02409-21. Epub 2022 May 16.
Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) is a crucial factor in bacterial virulence. The AS87_RS04190 protein was obviously missing from the secreted proteins of the T9SS mutant strain Yb2Δ. A bioinformatic analysis indicated that the AS87_RS04190 protein contains a T9SS C-terminal domain sequence and encodes a putative subtilisin-like serine protease (SspA). To determine the role of the putative SspA protein in R. anatipestifer pathogenesis and proteolysis, we constructed two strains with an mutation and complementation, respectively, and determined their median lethal doses, their bacterial loads in infected duck blood, and their adherence to and invasion of cells. Our results demonstrate that the SspA protein functions in bacterial virulence. It is also associated with the bacterial protease activity and has a conserved catalytic triad structure (Asp126, His158, and Ser410), which is necessary for protein function. The optimal reactive pH and temperature were determined to be 7.0 and 50°C, respectively, and and were determined to be 10.15 mM and 246.96 U/mg, respectively. The enzymatic activity of SspA is activated by Ca, Mg, and Mn and inhibited by Cu and EDTA. SspA degrades gelatin, fibrinogen, and bacitracin LL-37. These results demonstrate that SspA is an effector protein of T9SS and functions in R. anatipestifer virulence and its proteolysis of gelatin, fibrinogen, and bacitracin LL-37. In recent years, Riemerella anatipestifer T9SS has been reported to act as a virulence factor. However, the functions of the proteins secreted by R. anatipestifer T9SS are not entirely clear. In this study, a secreted subtilisin-like serine protease SspA was shown to be associated with R. anatipestifer virulence, host complement evasion, and degradation of gelatin, fibrinogen, and LL-37. The enzymatic activity of recombinant SspA was determined, and its and were 10.15 mM and 246.96 U/mg, respectively. Three conserved sites (Asp126, His158, and Ser410) are necessary for the protein's function. The median lethal dose of the -deleted mutant strain was reduced >10,000-fold, indicating that SspA is an important virulence factor. In summary, we demonstrate that the R. anatipestifer gene encodes an important T9SS effector, SspA, which plays an important role in bacterial virulence.
鸭传染性浆膜炎是一种主要的鸭败血性和渗出性疾病的病原。最近的研究表明，鸭疫里默氏杆菌（Riemerella anatipestifer）的 IX 型分泌系统（T9SS）是细菌毒力的一个关键因素。在 T9SS 突变株 Yb2Δ 的分泌蛋白中，明显缺失了 AS87_RS04190 蛋白。生物信息学分析表明，AS87_RS04190 蛋白含有 T9SS C 端结构域序列，并编码一个假定的枯草杆菌蛋白酶样丝氨酸蛋白酶（SspA）。为了确定假定的 SspA 蛋白在鸭疫里默氏杆菌发病机制和蛋白水解中的作用，我们分别构建了一个突变体和一个互补菌株，并确定了它们的半数致死剂量、感染鸭血液中的细菌载量以及它们对细胞的黏附和侵袭能力。我们的结果表明，SspA 蛋白在细菌毒力中起作用。它还与细菌蛋白酶活性有关，并具有保守的催化三联体结构（Asp126、His158 和 Ser410），这对于蛋白质功能是必需的。最佳反应 pH 和温度分别为 7.0 和 50°C，和分别为 10.15mM 和 246.96U/mg。SspA 的酶活性被 Ca、Mg 和 Mn 激活，被 Cu 和 EDTA 抑制。SspA 降解明胶、纤维蛋白原和杆菌肽 LL-37。这些结果表明，SspA 是 T9SS 的效应蛋白，在鸭疫里默氏杆菌的毒力及其对明胶、纤维蛋白原和杆菌肽 LL-37 的蛋白水解中起作用。近年来，鸭疫里默氏杆菌 T9SS 被报道为一种毒力因子。然而，鸭疫里默氏杆菌 T9SS 分泌的蛋白的功能并不完全清楚。在这项研究中，一种分泌的枯草杆菌蛋白酶样丝氨酸蛋白酶 SspA 被证明与鸭疫里默氏杆菌的毒力、宿主补体逃避以及明胶、纤维蛋白原和 LL-37 的降解有关。重组 SspA 的酶活性被测定，其和分别为 10.15mM 和 246.96U/mg。三个保守位点（Asp126、His158 和 Ser410）是蛋白质功能所必需的。缺失突变株的半数致死剂量降低了 >10000 倍，表明 SspA 是一个重要的毒力因子。总之，我们证明了鸭疫里默氏杆菌的基因编码了一个重要的 T9SS 效应子 SspA，它在细菌毒力中起着重要作用。
